Immunolocalization of a nuclear protein bound to the sphere organelle during oogenesis and embryogenesis inPleurodeles waltl

Summary The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.     

A maternal-effect mutation disturbs extracellular matrix organization in the early Pleurodeles waltl embryo

Summary In the early development of the urodele amphibian Pleurodeles waltl, a fibronectin-containing extracellular matrix underlies the inner face of the blastocoel roof. When gastrulation occurs, the fibronectin fibrils provide a suitable substrate for mesodermal-cell migration. Delay in morphogenetic movements of gastrulation has been described in embryos from mutant females (ac/ac) of Pleurodeles waltl. Studies of abnormal mutant gastrulae with fluorescent lectins and immunostaining for fibronectin reveal that they lack a normal matrix. The fibronectin-containing extracellular material always gives rise to a granular pattern without fibronectin-fibril formation. Fibronectin and ₅₁ syntheses occur normally in maternal-effect embryos. In vitro, mesodermal cells from early mutant gastrulae adhere and migrate on fibronectin-conditioned substrata.     

Three types of cutaneous glands in the skin of the salamandrid Pleurodeles waltl. A histological and ultrastructural study

Histological and ultrastructural investigations revealed three different multicellular skin gland types in the salamandrid Pleurodeles waltl. The mucous glands are small, with one layer of secretory cells surrounding a central lumen; they produce the viscous and slippery mucus film that has various functions in amphibians. The serous glands can be divided based on their histological and ultrastructural characters into the granular gland Type I (GGI) and the granular gland Type II (GGII). The first type (GGI) is moderately sized and distributed throughout the body surface, with higher concentrations in the parotoid and back regions. In contrast, the second type (GGII) is very large (for Pleurodeles) and was found only in the tail, with highest concentration in the tail dorsum. Both granular gland types contain mainly proteinaceous materials but differ in their morphological features including size, shape, cellular organization and vesicle distribution, vesicle size and vesicle shape. Both GGI and GGII are especially concentrated in body parts that are presented to an attacking predator and are hypothesized to produce repellent to poisonous substances to thwart potential aggressors. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Morphological variations in a tooth family through ontogeny in Pleurodeles waltl (Lissamphibia, Caudata)

Most nonmammalian species replace their teeth continuously (so-called polyphyodonty), which allows morphological and structural modifications to occur during ontogeny. We have chosen Pleurodeles waltl, a salamander easy to rear in the laboratory, as a model species to establish the morphological foundations necessary for future molecular approaches aiming to understand not only molecular processes involved in tooth development and replacement, but also their changes, notably during metamorphosis, that might usefully inform studies of modifications of tooth morphology during evolution. In order to determine when (in which developmental stage) and how (progressively or suddenly) tooth modifications take place during ontogeny, we concentrated our observations on a single tooth family, located at position I, closest to the symphysis on the left lower jaw. We monitored the development and replacement of the six first teeth in a large growth series ranging from 10-day-old embryos (tooth I1) to adult specimens (tooth I6), using light (LM), scanning (SEM), and transmission electron (TEM) microscopy. A timetable of the developmental and functional period is provided for the six teeth, and tooth development is compared in larvae and young adults. In P. waltl the first functional tooth is not replaced when the second generation tooth forms, in contrast to what occurs for the later generation teeth, leading to the presence of two functional teeth in a single position during the first 2 months of life. Larval tooth I1 shows dramatically different features when compared to adult tooth I6: a dividing zone has appeared between the dentin cone and the pedicel; the pulp cavity has enlarged, allowing accommodation of large blood vessels; the odontoblasts are well organized along the dentin surface; tubules have appeared in the dentin; and teeth have become bicuspidate. Most of these modifications take place progressively from one tooth generation to the next, but the change from monocuspid to bicuspid tooth occurs during the tooth I3 to tooth I4 transition at metamorphosis.     

Enameloid/enamel transition through successive tooth replacements in <Emphasis Type="Italic">Pleurodeles waltl</Emphasis> (Lissamphibia,Caudata)

Study of the evolutionary enameloid/enamel transition suffers from discontinuous data in the fossil record, although a developmental enameloid/enamel transition exists in living caudates, salamanders and newts. The timing and manner in which the enameloid/enamel transition is achieved during caudate ontogeny is of great interest, because the caudate situation could reflect events that have occurred during evolution. Using light and transmission electron microscopy, we have monitored the formation of the upper tooth region in six successive teeth of a tooth family (position I) in Pleurodeles waltl from late embryos to young adult. Enameloid has only been identified in embryonic tooth I₁ and in larval teeth I₂ and I₃. A thin layer of enamel is deposited later by ameloblasts on the enameloid surface of these teeth. From post-metamorphic juvenile onwards, teeth are covered with enamel only. The collagen-rich enameloid matrix is deposited by odontoblasts, which subsequently form dentin. Enameloid, like enamel, mineralizes and then matures but ameloblast participation in enameloid matrix deposition has not been established. From tooth I₁ to tooth I₃, the enameloid matrix becomes ever more dense and increasingly comes to resemble the dentin matrix, although it is still subjected to maturation. Our data suggest the absence of an enameloid/enamel transition and, instead, the occurrence of an enameloid/dentin transition, which seems to result from a progressive slowing down of odontoblast activity. As a consequence, the ameloblasts in post-metamorphic teeth appear to synthesize the enamel matrix earlier than in larval teeth.     

A study of cell interactions involved in Pleurodeles waltlii epidermal differentiation

Summary A polyclonal antibody (SP-2) has been produced, which recognizes antigens expressed in epidermal cells of Pleurodeles waltlii embryos. The antigens appear first at the end of gastrulation in the external surface of the embryo and are selectively expressed in ectodermally derived epidermal structures. Ectodermal commitment was investigated using cell cultures and blastocoel graft experiments. The four animal blastomeres of the 8-cell stage as well as the animal cap explants of the early gastrula stage cultured in vitro differentiate into epidermis, and SP-2 antigens are expressed. The expression of SP-2-defined antigens is inhibited both in vivo and in vitro by the inductive interaction of chordomesoderm. Once dissociated, ectodermal cells do not react with SP-2. Conversely, the aggregation of ectodermal cells may restore the expression of SP-2 antigens. Transplantation of animal cap explants or isolated ectodermal cells into the blastocoel of a host embryo at the early gastrula stage shows that only cells integrated into the epidermis express the marker antigens. When vegetal cells were dissociated from donor embryos before the mid-blastula stage and implanted into the blastocoel of host embryos at the early gastrula stage, their progeny were found in all germ layers, cells that were found in the host epidermis were stained with SP-2, whereas those contributing to mesoderm and endoderm were not. Thus the acquisition of cell polarity in epidermal differentiation and the organization of cells into epithelial structures are essential for SP-2-defined antigen expression.     

Application of local gene induction by infrared laser‐mediated microscope and temperature stimulator to amphibian regeneration study

Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three‐dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single‐cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR‐LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature‐controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR‐LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre‐LoxP system as well as to analyze gene function in a target region or cells with less off‐target effects in the study of amphibian regeneration.     

Isolation of highly polymorphic microsatellite loci for a species with a large genome size: sharp-ribbed salamander (Pleurodeles waltl)

Fourteen highly polymorphic microsatellite markers were developed and characterized for the sharp-ribbed salamander, Pleurodeles waltl. Isolating microsatellites with more than 12 single repeat type units was only successful for a tetranucleotide repeat (ATAG). Compared to microsatellite libraries constructed simultaneously for two anuran amphibian species, a greater number of primer pairs designed for P. waltl had to be discarded, due to consistent amplification problems. Low amplification success rate for P. waltl may be due to its larger genome size. Consequently, to avoid nonspecific binding and to increase amplification success, polymerase chain reaction programmes with touchdown cycles were used. For 14 microsatellite markers, amplification was successful and consistent with number of alleles and expected heterozygosity ranging from seven to 22 and from 0.79 to 0.94, respectively. All 14 microsatellite markers will be extremely useful for metapopulation studies of this unique amphibian species.     

Distribution of GABA-immunoreactive neurons in the brain of adult and developing salamanders (Pleurodeles waltli,Triturus alpestris)

The distribution of GABAergic neurons in brains of the family Salamandridae (Pleurodeles waltli, Triturus alpestris) has been investigated immunohistochemically with an antibody against gamma-aminobutyric acid (GABA). In adult animals, immunoreactive neurons, fibers, and terminals are abundantly labeled. In the telencephalon, pallial areas contain fewer GABAergic neurons and fibers than basal forebrain areas. The amygdalar complex and the habenulae have a complex pattern of GABA-immunoreactivity that is especially pronounced within the neuropil. The pretectal and basal optic systems are provided with GABAergic neurons, corroborating electrophysiological results. The dorsal thalamus and parts of the torus semicircularis are almost completely devoid of GABA-immunoreactive neurons. In the torus, magnocellular neurons known to project to the contralateral counterpart are distinctly GABA-immunoreactive. During ontogeny, GABAergic neurons arise early when the first reflexive movements occur after mechanical stimulation. At stage 28, cells are labeled initially near the nucleus of the medial longitudinal fasciculus, which is the first supraspinal tract to appear in ontogeny. At stage 30 (still before hatching), GABAergic neurons are found in the pretectum, immunoreactive neurons arising in the dorsal tegmentum slightly later. Both systems are known to mediate basic reflexes in gaze stabilization. The commissura posterior is GABAergic at early stages suggesting an important functional role in homonymous inhibition between both sides. Thus in salamanders, the neurotransmitter GABA displays a complex distribution, similar to that in other vertrebrates. This pattern emerges early in ontogeny.